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Food restriction has been recommended as an effective strategy for body weight loss. However, food restriction can alter biological rhythms and leads to physiological stress. However, relatively little is known about the physiological impact of different methods of food restriction.
Int J Med Sci 2018, Vol 15 Ivyspring International Publisher 1667 International Journal of Medical Sciences 2018; 15(14): 1667-1675 doi: 10.7150/ijms.28503 Research Paper Infrequent Feeding of Restricted Amounts of Food Induces Stress and Adipose Tissue Inflammation, Contributing to Impaired Glucose Metabolism Young-Sun Lee1, Changmi Lee1, Hee-Sook Jun1,2,3 Lee Gil Ya Cancer and Diabetes Institute, Gachon University, 7-45 Songdo-dong, Yeonsu-ku, Incheon 406-840 Korea College of Pharmacy and Gachon Institute of Pharmaceutical Science, Gachon University, 7-45 Songdo-dong, Yeonsu-ku, Incheon 406-840 Korea Gachon Medical Research Institute, Gil Hospital, Incheon, 405-760 Korea Corresponding author: Young-Sun Lee, Lee Gil Ya Cancer and Diabetes Institute, Gachon University, 7-45 Songdo-dong, Yeonsu-ku, Incheon 406-840 Korea Tel: 11-82-32-899-6217, Fax: 11-82-32-899-6232, E-mail: leeys@gachon.ac.kr © Ivyspring International Publisher This is an open access article distributed under the terms of the Creative Commons Attribution (CC BY-NC) license (https://creativecommons.org/licenses/by-nc/4.0/) See http://ivyspring.com/terms for full terms and conditions Received: 2018.07.13; Accepted: 2018.10.13; Published: 2018.11.05 Abstract Food restriction has been recommended as an effective strategy for body weight loss However, food restriction can alter biological rhythms and leads to physiological stress However, relatively little is known about the physiological impact of different methods of food restriction Therefore, we investigated whether different schedules of restricted food intake induce physiological stress and then contribute to glucose metabolism disorder C57BL/6 mice were fed a high fat diet (60% fat) for weeks and then randomly divided into three groups: the control group was continuously fed the high fat diet; the two food restriction groups were fed 50% of food consumed by the control mice with one group (FR1) being fed the full amount once a day and the other group (FR2) being fed the same total amount as FR1 twice a day for days We found increased body weight loss, the serum triglyceride levels, the expression of lipolysis-related genes, and serum corticosterone levels in the FR1 group compared with the FR2 group The immune cell population infiltrating the adipose tissue and the expression of monocyte chemoattractant protein (MCP-1) and toll-like receptor (TLR-4) mRNA were increased in the FR1 group compared with the control To determine whether long-term dietary manipulation is associated with metabolic disorders, mice were fed a restricted diet for days alternating with an unrestricted diet for the following days and this was repeated for weeks The alternating FR1 group showed impaired glucose tolerance compared with the alternating FR2 group These results indicate that infrequent feeding of restricted amounts of food could induce stress hormones, lipolysis, adipose tissue immune cell infiltration and inflammation, which in turn may promote glucose metabolism disorder Key words: Food restriction, stress, body weight, glucose metabolism, infrequent feeding Introduction Obesity prevalence has increased over the world Obesity has been identified as a risk factor for diabetes, cardiovascular disease, and many other diseases Weight loss is the first-line recommendation to reduce obesity Food restriction has been suggested as an effective therapeutic strategy for weight loss Many studies have shown that food restriction can alter biological rhythms [1-3] and leads to physiological stress [4] Thus, proper food quantity and frequency are crucial for maintaining a healthy condition [5] Stressful events are known to activate the hypothalamo-pituitary-adrenocortical axis, which results in increased plasma concentration of glucocorticoids, the “stress hormones” [6] The effects of glucocorticoids on body weight and lipid metabolism are controversial: some studies show that glucocorticoids reduce body weight and stimulate http://www.medsci.org Int J Med Sci 2018, Vol 15 lipolysis [7, 8], while others show an increase of body weight and lipogenesis [9, 10] This difference may depend on the glucocorticoid concentrations and duration of exposure [11] In addition, glucocorticoid treatments can induce insulin resistance in healthy individuals [12, 13] Obesity is associated with the accumulation of immune cells in adipose tissue, which contributes to the development of metabolic disorders [14, 15] Interestingly, weight loss is also associated with recruitment of immune cells in adipose tissue Fasting or pharmacologically induced lipolysis increases adipose tissue macrophage (ATM) accumulation in adipose tissue [16, 17] Inflammation and metabolic disease have been shown to be linked, and pro-inflammatory factors are involved in the pathogenesis of type diabetes [18] Although food restriction is strongly recommended to reduce body weight, relatively little is known about the physiological impact of different methods of food restriction Thus, we investigated whether different schedules of restricted food intake induce stress hormones and infiltration of immune cells in adipose tissue, contributing to glucose metabolic disorder Our data showed that infrequent feeding of restricted food amounts, as compared with more frequent feedings of the same amount of restricted food, induced stress, lipolysis, and accumulation of adipose tissue immune cells in mice, which may contribute to disorders of glucose metabolism Materials and methods Animals C57BL/6 mice were obtained from the Orient Bio INC (Gyeonggi, Korea) and maintained in specific pathogen-free conditions at the animal facility at Gachon University under a 12 h light:12 h dark photoperiod Animals were freely fed with a high fat diet containing 60% lipid (Research Diets Inc, New Brunswick, NJ) for weeks (diet-induced obesity (DIO) mice) and then randomly divided into three groups: the control group was continuously fed the 60% high fat diet; the two food restriction groups were fed 50% of the mean amount of food consumed by the control mice with one group being fed that amount (50%) once a day (FR1; fed at 9:00 AM) and the other group being fed the same amount divided into two portions (2 × 25%) given twice a day (FR2; fed at 9:00 AM and 9:00 PM) for days To examine the effects of long-term, dietary restriction, DIO mice were given restricted food as for the FR1 and FR2 protocols for days, and then food was unrestricted for the following days The alternating 1668 restricted/unrestricted diet was given for a total of weeks All animal experiments were carried out under a protocol approved by the Institutional Animal Care and Use Committee at the Gachon University Measurement of body weight Body weight was measured before restriction and 1, 2, and days thereafter food Histological staining of adipose tissue Mice were sacrificed at days after food restriction, and visceral adipose tissue was removed, fixed in 10% formalin, and embedded in paraffin To detect histological changes in the adipose tissue, paraffin-embedded sections were stained with hematoxylin and eosin Isolation of stromal vascular cells and adipocyte infiltrated immune cells Visceral adipose tissue was removed, cut with scissors into small pieces, and digested with mg/ml collagenase type (Sigma, NY, USA) for 30 at 37°C Stromal vascular cells were collected as a pellet after centrifugation at 1500 rpm for Immune cells infiltrating the adipose tissue were isolated by Ficoll density gradient centrifugation [19, 20] Real-time quantitative PCR (RT-qPCR) Total RNA was isolated from the adipose tissue or hypothalamus of food-restricted mice or adipose tissue from corticosterone-treated DIO mice, and cDNA was synthesized using the PrimeScript first-strand cDNA synthesis kit (TaKaRa Bio, Inc., Otsu, Japan) PCR was carried out in a 7900HT fast real-time PCR system (Applied Biosystems, Carlsbad, CA) at 95°C for 10 min, followed by 40 cycles at 95°C for 15 s, 60°C for As an internal control, cyclophilin mRNA was amplified The sequences of the primer pairs are shown in Supplementary Table The relative copy number was calculated using the threshold crossing point (Ct) as calculated by the 7900HT fast real-time PCR software combined with the delta delta Ct calculations Triglyceride and corticosterone analysis After days of food restriction, mice were sacrificed between 8-10 AM., and blood samples were collected Serum triglyceride and corticosterone levels were measured by a triglyceride assay kit (Asan Pharmaceutical Co Ltd., Seoul, Korea) and corticosterone ELISA assay kit (Enzo Life Sciences, Plymoth meeting, PA, USA) 3T3-L1 cell differentiation and treatment 3T3-L1 pre-adipocytes were cultured for days http://www.medsci.org Int J Med Sci 2018, Vol 15 in 6-well plates (1.0 × 105 cells per well) at 37°C in Dulbecco’s modified Eagle’s medium containing 1% penicillin–streptomycin solution (Gibco, Gaithersburg, MD) and 10% FBS The cells were then induced to differentiate in medium containing μM dexamethasone, μg/ml insulin and 0.5 mM 3-isobutyl-1-methylxantine as described previously [21, 22] Differentiated 3T3-L1 adipocytes were treated with various doses of corticosterone for 24 h Corticosterone treatment via an osmotic minipump Male C57BL/6 mice were fed a high-fat diet (60% energy by fat) for weeks Animals were treated either with corticosterone (15 μg h-1; Sigma) via an osmotic minipump (Alzet Model 1003D) or PBS for days Glucose and insulin tolerance tests After days of food restriction or weeks of the alternating restricted/non-restricted diet, mice were not fed for h, and then a glucose solution (2 g kg-1 body weight) was injected intraperitoneally Blood glucose levels were measured using a glucometer at 0, 30, 60, and 90 after glucose injection For insulin tolerance tests, mice were not fed for h and were 1669 injected with insulin (2 units kg-1 body weight, i.p.), and blood glucose levels were measured using a glucometer at 0, 30, 60, and 90 after insulin injection Statistical analysis Data are presented as means ±SEM Statistical significance was analyzed by unpaired Student’s t-test for comparison of two groups or ANOVA followed by Fisher’s protected least significant difference test for multiple groups P